The present invention relates generally to the use of polysaccharides containing arabinose, galactose and/or hexuronic acid in promoting in vivo and in vitro survival and improved function of sperm, oocytes, and embryos.
In nature, fertilization occurs by sperm cells being deposited into the female of warm-blooded animal species (including humans) and then binding to and fusing with an oocyte. This fertilized oocyte then divides to form an embryo. Over the last several decades, the use of assisted reproduction techniques has allowed scientists and clinicians to intervene in these events to treat poor fertility in some individuals or to store sperm, oocytes or embryos for use at other locations or times. The procedures utilized in these cases include: washing a sperm sample to separate out the sperm-rich fraction from non-sperm components of a sample such as seminal plasma or debris; further isolating the healthy, motile (swimming) sperm from dead sperm or from white blood cells in an ejaculate; freezing or refrigerating of sperm (storage) for use at a later date or for shipping to females at differing locations; extending or diluting sperm for culture in diagnostic testing or for use in therapeutic interventions such as in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI); culturing or freezing oocytes from the female for use in in vitro fertilization; and culturing or freezing of embryos prior to transfer back to a female in order to establish a pregnancy.
At each step of the way, in vitro intervention decreases the normal survival and function of sperm, oocytes, and embryos. Much research has been dedicated towards improving these procedures; however, overall success remains limited. For example,  less than 20% of IVF attempts result in the birth of a child. Additionally, only half or less of sperm cells routinely survive the freezing process, such that pregnancy rates with frozen sperm from donors average between 10 and 20%. Oocytes and embryos also show significantly disrupted function after culture or freezing. Specifically, human oocytes survive the freezing process at very low levels. Thus, in spite of several decades of work, much room remains for improvement in the field of assisted reproduction technologies and especially in gamete and embryo handling, culture, and storage.
One common procedure used in sperm collection is washing sperm cells. Washing sperm prior to its use in assisted reproduction technologies is important for a variety of reasons. An ejaculate contains seminal plasma in addition to sperm cells, and the sugars and proteins in seminal plasma can be toxic to sperm cells after ejaculation. Also, sperm samples that have been frozen contain cryopreservation media which needs to be washed from the sperm cells prior to insemination in the female of some species, particularly birds and women. For all species, cryopreservative media cause lipid membrane peroxidation (LPO) and degeneration of the sperm after thawing. Generally, washing involves centrifuging a sample of semen or thawed sperm through a diluting wash media, which allows collection of a sperm-rich pellet. Although a very common procedure, centrifugation itself can cause sperm lipid peroxidation and membrane breakdown.
After a sperm wash process, or in place of it, a specific procedure for the isolation of the motile sperm from a sample may be done. An ejaculate contains dead and dying sperm that release enzymes that can damage the live, motile sperm. In addition, an ejaculate contains white blood cells, red blood cells, and bacteria which are also toxic to the healthy sperm in an ejaculate. Sperm isolations involve separating out the live, healthy, and motile sperm for use in diagnostic or therapeutic procedures. Generally, sperm are isolated by allowing the motile sperm to swim away from the dead sperm and debris (sperm swim-up), by centrifuging the sperm through a density gradient, or by passing the sperm through a column that binds the dead sperm and debris. Each of these techniques has its own disadvantages. Swim-up only recovers low sperm numbers, and it requires a long culture period. Current centrifugation gradient reagents are generally toxic to sperm, such that an added wash step is necessary to remove the gradient solution from the sperm sample. Column methods have poor selectivity for motile sperm and do not always result in good recovery of sperm numbers from a full ejaculate.
Once sperm have been washed or isolated, they are then extended (or diluted) in culture or holding media for a variety of uses. Existing sperm culture techniques result in losses of motile sperm and also damage sperm DNA over time in culture. Although sperm survive for days in the females of most species, sperm survival in culture is typically only half as long as that seen in vivo, and sperm from males with poor quality ejaculates may survive for even shorter time periods in culture. Much of this damage is due to lipid peroxidation of the membrane and DNA or chromatin breakdown. Sperm are extended in media for use in sperm analysis and diagnostic tests; assisted reproduction technologies, such as IVF, gamete intrafallopian transfer, or ICSI; insemination into the female; and holding prior to cryopreservation. Each of these uses for extended or diluted sperm requires a somewhat different formulation of basal medium; however, in all cases sperm survival is suboptimal outside of the female reproductive tract.
Likewise, oocytes and embryos often develop abnormally (e.g., chromosome number, cytoskeleton formation) in culture compared to in vivo conditions. Additionally, current culture methods utilize high doses of animal proteins, like serum, which may result in an oversized fetus and perinatal complication for the offspring.
Some of the difficulties in assisted reproduction technologies can be overcome by coculturing sperm, oocytes and embryos with cell feeder layers. However, cocultures are of variable quality and variable reliability and add the risk of pathogen transfer from the feeder cells to the gametes or embryos that are to be transferred back to living animals or humans.
Storage of sperm is of widespread importance in commercial animal breeding programs, human sperm donor programs and in dealing with some disease states. For example, sperm samples may be frozen for men who have been diagnosed with cancer or other diseases that may eventually interfere with sperm production. Freezing and storage of sperm is critical in the area of preservation of endangered species. Many of these species have semen which does not freeze well under existing methods. In standard animal husbandry, artificial insemination (AI) with frozen bull sperm is used in 85% of dairy cows. Because most commercial turkeys have become too heavy to mate naturally, AI is required on almost all turkey farms. Approximately six million turkey hens are inseminated each week in the United States. However, existing methods of storing collected turkey sperm cannot support sperm survival for even the several hours required to transport semen between farms, much less for long-term freezing. This limits the ability to store or transport genetic material to improve production. Human donor AI is also used for couples with severe male infertility; however, pregnancy rates with donor semen in people is only a quarter of that found with natural reproduction. Furthermore, surgical insemination may be required.
Current techniques for freezing sperm from all species result in membrane damage and subsequent death of about half of the sperm cells in a sample. Much of this damage occurs by reactive oxygen species causing lipid peroxidation of the sperm membrane. Despite these widespread and serious problems, the state of the art and protocols for this field have changed very little in the last 15 years. In light of the increasing use of frozen sperm in a variety of settings, a new method of freezing or storing sperm would offer a major breakthrough for human fertility specialists, animal producers, and conservation specialists.
Freezing oocytes and embryos is also important for preserving genetic material from endangered species, increasing offspring production from valuable livestock individuals, or for retaining embryos for infertile couples prior to transfer. Current methods of freezing oocytes and embryos are less than optimal with decreased development potential seen. In fact, human oocytes are rarely successfully frozen, necessitating placing multiple embryos into a woman""s uterus which increases the number of dangerous and high risk multiple pregnancies. In addition, IVF embryos or genetically altered embryos from all species, such as those obtained after gene therapy, have very poor post-freezing survival rates with existing freezing media. This includes cloned embryos and embryos derived from embryonic stem cells (ESC).
For couples with fertility problems, an alternative course to the assisted reproduction techniques described above for improving the chance of conception is to have multiple, timed events of coitus during oocyte ovulation. For many of these couples, the emotional stress of infertility and the necessity of timed coitus month after month can lead to the need for artificial lubrication during intercourse. However, most commercially available lubricants are spermicidal, as is saliva, so that infertile couples are often instructed by their physician to not utilize any lubricant products during intercourse. In addition, many aspects of reproductive medicine in both humans and animals would be enhanced by the use of non-spermicidal lubrication during procedures such as manual sperm sample collection, artificial insemination, and uterine catheterization. The lubricant products on the market are not acceptable for use in these situations because of their spermicidal properties.
Following deposition in the female, sperm must penetrate the cervical mucus of the female and swim to the oviducts (Fallopian tubes) where they remain until ovulation and fertilization occurs. Sperm that are compromised may not be able to swim through this mucus and are thus not available for the fertilization process, limiting the fertility of the male. Furthermore, sperm that are slow to enter the cervical mucus are left in contact with the vaginal mucosa which is acidic and can inactivate sperm within several hours. Sperm with impaired fertilization potential include those that have been frozen, those where the male has antibodies in his semen that weaken the sperm, or sperm that have abnormal motion or shapes. Therapeutic options for treating male factor infertility, which accounts for 60% of infertility cases, are currently very limited and often end up utilizing very expensive intervention techniques, such as ICSI in which a single sperm is injected into an egg. As well, an increased incidence in genetic and/or birth defects have been reported for offspring from such sperm injection techniques. A product that improves sperm survival, motility and mucus penetration after ejaculation or insemination in the female could increase the number of sperm available in the oviduct for fertilization and thus could improve the chances of conception occurring without invasive intervention.
The present invention provides a variety of compositions that are non-toxic to sperm, oocytes or embryos, which additionally improve their function and survival during in vitro handling and which improve sperm function for use by couples trying to conceive naturally, as well as for use in a variety of assisted reproduction techniques in humans and animals. The present invention further provides other related advantages.
The present invention provides methods and compositions for improving the function of germ cells (sperm and oocytes), and embryos both in vivo and in vitro.
Within one aspect, methods for isolation of motile sperm having improved function are provided comprising contacting a sample containing sperm with a solution comprising a polysaccharide containing arabinose, galactose and/or hexuronic acid (PCAGH) to form a mixture, wherein the PCAGH is not arabinogalactan, and then removing the wash solution. This mixture is subjected to conditions sufficient to separate the motile sperm from the rest of the sample, thereby isolating the sperm with improved function. In a related aspect, methods for washing sperm to remove the nonsperm portion of a sample and to obtain sperm with improved function are provided comprising contacting a sample containing sperm with a solution comprising a polysaccharide containing arabinose, galactose and/or hexuronic acid, wherein the PCAGH is not arabinogalactan, and removing the wash solution. Within certain embodiments, the polysaccharide is pectin, arabic acid, gum arabic, gum ghatti, gum karaya, gum guar, galactopyranosylarabinose, galacturonic acid, gum locust bean, gum tragacanth, carrageenan, or derivatives thereof. Within another embodiment, the sample is semen. Within yet other embodiments, the sample is obtained from human, bovine, canine, equine, porcine, ovine, avian, rodent or exotic species. In certain embodiments, it may also include other density gradient compounds, such as dextran, iodixanol, sucrose polymers, nycodenz, or polyvinylpyrolidine coated silica (Percoll). In other embodiments, the solution comprises a balanced salt solution and a macromolecule.
Within another aspect, a sperm wash medium is provided comprising a polysaccharide containing arabinose, galactose and/or hexuronic acid (PCAGH) and a macromolecule wherein the PCAGH is not arabinogalactan. The polysaccharide is present at a concentration sufficient to improve sperm function at 1-50%. In certain embodiments, the macromolecule is gelatin, bovine serum albumin, human serum albumin, egg yolk, oviductin, polyvinyl alcohol, hyaluronic acid, gelatin, catalase, or casein. Generally, the solution further comprises a balanced salt solution.
Within a related aspect, a medium for the isolation of motile sperm from a sample is comprised of a PCAGH at 0.01-5% and a density gradient compound for centrifugation isolation, or a macromolecule for swim-up separation.
Within another related aspect, an extending medium for sperm is provided comprising a PCAGH in a solution at a concentration sufficient to improve sperm function.
In another aspect, a non-spermicidal lubricant for increasing fertilization potential in animals is provided comprising a non-spermicidal lubricious compound and a polysaccharide containing arabinose, galactose and/or hexuronic acid (PCAGH). Within certain embodiments, the lubricious compound comprises glycerine, methylcellulose, propylene glycol, plant oils, or petroleum jelly, or a combination of glycerin and petroleum jelly, or a combination of polyethylene oxide, sodium carboxypolymethylene and methylparaben. Within other embodiments, the polysaccharide is pectin, arabinogalactan, arabic acid, gum arabic, gum ghatti, gum karaya, gum guar, galactopyranosylarabinose, galacturonic acid, gum locust bean, gum tragacanth, carrageenan, or a derivative thereof. The lubricant may be used in vivo by administration or placement in a vagina prior to coitus or artificial insemination, or used during semen collection, such as by applying the lubricant to a penis prior to ejaculation into a receptacle or collecting sperm into a receptacle containing the lubricant. In a related aspect, the lubricant is used to lubricate medical devices or a hand prior to reproductive procedures.
In yet other aspects of the subject invention, methods for increasing the survival of sperm, oocyte, embryo and embryonic stem cells in vitro are provided comprising contacting a sample containing one of the cell types with a medium acceptable to the cell and including a polysaccharide containing arabinose, galactose and/or hexuronic acid (PCAGH). Within certain preferred embodiments, the medium is a balanced salt solution medium. Within other embodiments, the medium further comprises a macromolecule, such as blood serum, synthetic serum supplements, bovine serum albumin, human serum albumin, oviductin, superoxide dismutase, vitamin E, gelatin, polyvinyl alcohol, hyaluronic acid, catalase, chondroitin sulfate, heparin, egg yolk, skim milk, casein, melanin, hormone or growth factors. With other embodiments, the medium also comprises a sperm stimulant. Sperm stimulants include caffeine, follicular fluid, oxytocin, kallikrien, prostaglandins, thymus extract, pentoxyfilline, deoxyadenosine, inositol, platelet activating factor, hypotaurine, or mercaptoethanol.
Within yet other aspects, methods for reducing the loss of functional sperm, reducing the cellular damage to an oocyte, and reducing the cellular damage to an embryo or embryonic stem cell (ESC), resulting from the storage of the cells in a refrigerated, frozen or vitrified state are provided. More specifically, a polysaccharide containing arabinose, galactose and/or hexuronic acid (PCAGH) and a sample containing sperm, oocyte, or embryo, are combined wherein the polysaccharide is in an amount effective to reduce the loss or damage and the sample is then stored in a refrigerated, frozen or vitrified state. Within certain preferred embodiments, an additional cryoprotective compound is added. Within a related aspect, a medium for storing sperm, oocytes, or embryos is provided comprising a balanced salt solution and a polysaccharide containing arabinose, galactose and/or hexuronic acid.
These and other aspects of the invention will become evident upon reference to the following detailed description and attached drawings. In addition, various references are set forth below which describe in more detail certain procedures or compositions. Each of these references are incorporated herein by reference in their entirety as if each were individually noted for incorporation.